Dong Z, Patel Y, Saikumar P, Weinberg J M, Venkatachalam M A
Department of Pathology, University of Texas Health Science Center at San Antonio, 78284-7750, USA.
Lab Invest. 1998 Jun;78(6):657-68.
Studies during the past decade have led to the recognition of a fundamental, widely expressed mechanism of structural damage in energy-deprived cells, which is suppressed by physiologic levels of glycine and is independent of Ca2+ availability or alterations of cytosolic free Ca2+. To gain insight into this process, Madin-Darby canine kidney (MDCK) cells were depleted of adenosine triphosphate (ATP) by a mitochondrial uncoupler in glucose-free medium, and intracellular free Ca2+ was clamped at 100 nM to avoid calcium cytotoxicity. Although the ATP-depleted cells swelled and blebbed and their plasma membranes appeared to be under tension, they nevertheless became permeable to macromolecules. The plasma membranes of these cells retained structural continuity, as determined by morphologic observations, and confocal microscopy of a plasma membrane protein label (Biotin: Ultra Avidin-Texas Red) and a lipid label (NBD-sphingomyelin). Using fluoresceinated dextrans of graded molecular size, membrane permselectivity was examined noninvasively by confocal microscopy. Measured as inside/outside ratios of fluorescence intensity, the permeability indices showed progressively greater restriction to diffusion of increasingly larger dextran molecules across plasma membranes, with sharp break-points between 70,000 and 145,000 daltons (d). The results indicated that the membranes behaved as if they were perforated by water-filled channels or "pores," with size-exclusion limits of molecular dimensions. The membrane defects evolved from small pores permeable only to propidium iodide (668 d) and the smallest dextran (4,000 d), before enlarging with time to become permeable to larger dextrans. Inclusion of glycine during ATP depletion did not affect cell swelling or blebbing but completely prevented the development of permeability defects. Treatment of cells before ATP depletion with a membrane-impermeant homobifunctional "nearest neighbor" cross-linking agent, 3,3' dithiobis(sulfosuccinimidylpropionate), suppressed the development of permeability defects, even in the absence of glycine. These observations suggest that the cellular abnormality that is suppressed by glycine involves rearrangement of plasma membrane proteins to form water-filled pores large enough to leak macromolecules.
过去十年间的研究已使人们认识到一种在能量缺乏细胞中普遍存在的基本结构损伤机制，该机制受生理水平甘氨酸的抑制，且与细胞外钙离子浓度或胞质游离钙离子的变化无关。为深入了解这一过程，在无葡萄糖培养基中，用线粒体解偶联剂使麦迪逊-达比犬肾（MDCK）细胞内的三磷酸腺苷（ATP）耗竭，并将细胞内游离钙离子浓度钳制在100 nM以避免钙细胞毒性。尽管ATP耗竭的细胞出现肿胀和出泡现象，且其质膜似乎处于张力状态，但它们对大分子仍具有通透性。通过形态学观察以及对质膜蛋白标记物（生物素：超抗生物素蛋白-德克萨斯红）和脂质标记物（NBD-鞘磷脂）进行共聚焦显微镜观察，确定这些细胞的质膜保持结构连续性。使用不同分子大小的荧光葡聚糖，通过共聚焦显微镜对膜的通透选择性进行无创检测。以荧光强度的胞内/胞外比值衡量，通透性指数显示，随着葡聚糖分子越来越大，其跨质膜扩散受到的限制逐渐增强，在70,000至145,000道尔顿（d）之间有明显的转折点。结果表明，这些膜的行为就好像被充满水的通道或“孔”穿透，具有分子尺寸的排阻极限。膜缺陷最初表现为仅对碘化丙啶（668 d）和最小的葡聚糖（4,000 d）可通透的小孔，随后随着时间推移扩大，变得对更大的葡聚糖也具有通透性。在ATP耗竭期间加入甘氨酸并不影响细胞肿胀或出泡，但完全阻止了通透性缺陷的发展。即使在没有甘氨酸的情况下，在ATP耗竭前用一种不能透过膜的同型双功能“近邻”交联剂3,3'-二硫代双（磺基琥珀酰亚胺丙酸酯）处理细胞，也能抑制通透性缺陷的发展。这些观察结果表明，被甘氨酸抑制的细胞异常涉及质膜蛋白重排，形成足以使大分子渗漏的充满水的孔。
